Insight into the uncoupling mechanism of sarcoplasmic reticulum ATPase using the phosphorylating substrate UTP |
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Authors: | Fortea M I Soler F Fernandez-Belda F |
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Institution: | Departamento de Bioquimica y Biologia Molecular A, Edificio de Veterinaria, Universidad de Murcia en Espinardo, 30071 Murcia, Spain. |
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Abstract: | Ca(2+) transport and UTP hydrolysis catalyzed by sarcoplasmic reticulum Ca(2+)-ATPase from skeletal muscle was studied. A passive Ca(2+) load inside microsomal vesicles clearly decreased the net uptake rate and the final accumulation of Ca(2+) but not the UTP hydrolysis rate, causing energy uncoupling. In the absence of passive leak, the Ca(2+)/P(i) coupling ratio was 0.7-0.8. UTP hydrolysis did not maintain a rapid component of Ca(2+) exchange between the cytoplasmic and lumenal compartments as occurs with ATP. The uncoupling process in the presence of UTP is associated with: (i) the absence of a steady state accumulation of ADP-insensitive phosphoenzyme; (ii) the cytoplasmic dissociation of Ca(2+) bound to the ADP-sensitive phosphoenzyme; and (iii) the absence of enzyme inhibition by cyclopiazonic acid. All these characteristics confirm the lack of enzyme conformations with low Ca(2+) affinity and point to the existence of an uncoupling mechanism mediated by a phosphorylated form of the enzyme. Suboptimal coupling values can be explained in molecular terms by the proposed functional model. |
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